Skip to main content
Download PDF

Genome-wide characterization of COMT family and regulatory role of CsCOMT19 in melatonin synthesis in Camellia sinensis

BMC Plant Biology volume 24, Article number: 51 (2024) Cite this article

Abstract

Background

Caffeic acid O-methyltransferase (COMT) is a key enzyme that regulates melatonin synthesis and is involved in regulating the growth, development, and response to abiotic stress in plants. Tea plant is a popular beverage consumed worldwide, has been used for centuries for its medicinal properties, including its ability to reduce inflammation, improve digestion, and boost immune function. By analyzing genetic variation within the COMT family, while helping tea plants resist adversity, it is also possible to gain a deeper understanding of how different tea varieties produce and metabolize catechins, then be used to develop new tea cultivars with desired flavor profiles and health benefits.

Results

In this study, a total of 25 CsCOMT genes were identified based on the high-quality tea (Camellia sinensis) plant genome database. Phylogenetic tree analysis of CsCOMTs with COMTs from other species showed that COMTs divided into four subfamilies (Class I, II, III, IV), and CsCOMTs was distributed in Class I, Class II, Class III. CsCOMTs not only undergoes large-scale gene recombination in pairs internally in tea plant, but also shares 2 and 7 collinear genes with Arabidopsis thaliana and poplar (Populus trichocarpa), respectively. The promoter region of CsCOMTs was found to be rich in cis-acting elements associated with plant growth and stress response. By analyzing the previously transcriptome data, it was found that some members of CsCOMT family exhibited significant tissue-specific expression and differential expression under different stress treatments. Subsequently, we selected six CsCOMTs to further validated their expression levels in different tissues organ using qRT-PCR. In addition, we silenced the CsCOMT19 through virus-induced gene silencing (VIGS) method and found that CsCOMT19 positively regulates the synthesis of melatonin in tea plant.

Conclusion

These results will contribute to the understanding the functions of CsCOMT gene family and provide valuable information for further research on the role of CsCOMT genes in regulating tea plant growth, development, and response to abiotic stress.

Peer Review reports

Background

Melatonin, chemically named N-acetyl-5-methoxytryptamine, is a highly conserved small-molecule tryptophan indole derivative. Melatonin and its metabolites have attracted attention due to their ability to continuously scavenge reactive oxygen species (ROS) or reactive nitrogen species (RNS) [1]. Study showed that one molecule of melatonin can scavenge up to ten ROS or RNS molecules, thus even lower concentrations of melatonin can effectively protect organisms against oxidative stress [2, 3]. Exogenous application of melatonin could slow down senescence caused by various abiotic stresses and regulate physiological processes such as circadian rhythm, explant growth and flowering, and seed germination as a multifunctional signaling molecule [4,5,6,7,8,9].

In recent years, research has found that plant melatonin is mainly regulated by six different enzymes, including tryptophan decarboxylase (TDC), tryptophan hydroxylase (TPH), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), acetylserotonin methyl transferase (ASMT), and caffeic acid O-methyltransferase (COMT) [4]. Among them, COMT and ASMT that belong to the O-methyltransferase family can methylate phenylpropane compounds, flavonoids, and alkaloids [10]. COMT has been found in various species such as Arabidopsis thaliana [11], rice (Oryza sativa) [12], longan (Dimocarpus longan) [13], etc. COMTs catalyze N-acetyl serotonin into melatonin [10, 14]. The overexpression of them also can help plant grow [15]. Sorghum bicolor COMT can be involved in tricin biosynthesis methylated the flavones luteolin and selgin [16]. The expression of COMT4 in aspen can change the structure of lignin, which increase the crosslinking of condensed lignin subunits by G-units [17]. However, little information is available about the COMT genes and their functions in tea plants.

Tea plant (Camellia sinensis) is an important economic crop, and non-alcoholic beverages made from its buds and leaves are popular worldwide [18, 19]. Tea contains many health benefits such as aiding digestion, antibacterial effects, reducing blood pressure, sugar and cholesterol, antioxidant effects, and etc. due to its high content of bioactive catechins, vitamins, polyphenols, flavonoids, and medicinal properties [20,21,22]. As a plant that thrives in warm and humid environments, tea plants are often subjected to various external stresses during their growth process, such as drought and low temperature stress, pest and disease infestations, which can significantly reduce the growth, development, quality, and yield of tea leaves. Due to environmental changes, problems caused by abiotic stresses such as drought and low temperature have become increasingly prominent, leading to significant reductions in tea production and quality and causing enormous economic losses [23]. Therefore, exploration of the mechanisms underlying tea plant stress tolerance and in-depth mining of stress-related genes are of great theoretical and practical significance.

In this study, we conducted a whole-genome identification of the CsCOMT family in tea plant using bioinformatics methods based on the high-quality tea plant genome database. A total of 25 CsCOMT genes were identified, and the physicochemical properties, gene structures, and subcellular localization were predicted. The structural characteristics and evolutionary traits of the tea plant CsCOMT family were clarified. Simultaneously, we also analyzed the expression patterns of 25 CsCOMT genes in different tissues and under stress based on the transcriptome data. In addition, the virus-induced gene silencing (VIGS) approach was used to investigate the effect of the CsCOMT19 on melatonin content. This study aims to provide reference for revealing the potential functions of CsCOMT family members in tea plants.

Results

Identification of the CsCOMT family genes in tea plant

Using the amino acid sequence of Arabidopsis COMT as a BLAST query, we identified 25 candidate tea plant COMT genes (CsCOMTs) from the tea plant genome database. Then, these candidate genes were submitted to online websites for structural validation of conserved domains through two rounds of comparison search. Finally, all 25 CsCOMT genes were confirmed to belong to the COMT family and named based on their homology with Arabidopsis COMT and their position on chromosomes.

Through the analysis of the physicochemical properties of members of the CsCOMT gene family, it was found that the CsCOMT proteins had an amino acid range of 186 (CsCOMT25) to 393 (CsCOMT13); the relative molecular weight was between 20.73 (CsCOMT25) to 43.80 kD (CsCOMT13); the theoretical isoelectric point (pI) ranged from 4.94 (CsCOMT1) to 6.49 (CsCOMT25). It was found that all of 25 CsCOMT proteins have a pI < 7, indicating that most CsCOMT proteins are rich in acidic amino acids. Most subcellular localizations of CsCOMT family members were in chloroplasts, among which CsCOMT5 was located in mitochondria, CsCOMT13 was located in both chloroplasts and mitochondria, and CsCOMT25 was located in the nucleus (Table 1).

Table 1 Information of CsCOMT genes identified in C. sinensis
Full size table

Phylogenetic analysis and conserved motifs of CsCOMTs

To clarify the classification of the CsCOMT family members, multiple sequence alignments were performed using CsCOMTs and COMTs from Arabidopsis, rice, and poplar. The results showed that CsCOMTs had high homology with COMTs from other species. According to the analysis of the evolutionary relationships among tea plants, Arabidopsis, rice, and poplar, the members of the COMT family were divided into four subfamilies (Fig. 1, Fig. S1). The members of the tea tree CsCOMT family are distributed in Class I, Class III, and Class V. Among them, CsCOMT has the highest number of members in Class I (12 members) (Fig. S2).

Fig. 1
figure 1

Phylogenetic analysis of COMTs identified in tea plants, Arabidopsis, poplar, and rice. Different colors represent different groups, and different abbreviations represent COMTs from tea plant (CsCOMT), Arabidopsis (AtCOMT), poplar (PtCOMT), and rice (OsCOMT)

Full size image

Further analyzed the conserved motifs of CsCOMT family members, and the results showed that CsCOMT family members contained a large number of co-existing motifs, including motif 3 (Fig. 2, Fig. S3). The type of conserved motifs in each subfamily were basically the same, indicating that the same subfamily was composed of similar conserved structural domains and may have similar biological functions.

Fig. 2
figure 2

The conserved motifs of CsCOMTs

Full size image

Chromosomal localization and gene structure of CsCOMT members

The members of CsCOMT family were most distributed on chromosome 5, with seven members, while chromosomes 1, 8, and 9 were the least distributed, with one member on each chromosome (Fig. 3, Table S1). In order to gain a deeper understanding of the CsCOMT family genes function, the structure of CsCOMTs were analyzed. By analyzing the sequence composition of the CsCOMT gene, it was found that all CsCOMTs in tea plants contain introns. From the distribution of UTR, we can see that the UTR of CsCOMT gene family is relatively small (Fig. 4).

Fig. 3
figure 3

The chromosome localization of CsCOMT genes

Full size image
Fig. 4
figure 4

Structures of the 25 CsCOMT genes

Full size image

Further analysis of the secondary structure of the CsCOMT family members in tea plants revealed that CsCOMT is mainly composed of helix, followed by coil, and finally strand (Fig. 5). Overall, these three structures are intertwined. Subsequently, protein tertiary structure prediction was performed, and it was found that the tertiary structures of CsCOMTs was mostly single-cluster shape, expect that CsCOMT10 showed a double-cluster distribution. In addition, CsCOMT1, CsCOMT2, CsCOMT3, CsCOMT4, and CsCOMT5 exhibited different shapes from other family members (Fig. 6).

Fig. 5
figure 5

Secondary structure of CsCOMT family members

Full size image
Fig. 6
figure 6

Tertiary structure of CsCOMT family members

Full size image

Collinearity analysis

Through collinearity analysis, it was found that there was a large segment duplication among members of the CsCOMT family, including CsCOMT17 and CsCOMT18, CsCOMT19 and CsCOMT9. Subsequently, we conducted a large segment recombination analysis between tea plant and Arabidopsis and found that CsCOMT17, CsCOMT18, and AtCOMT14 underwent large segment recombination. In addition, many large segment recombination events were also observed between tea plant and poplar, including CsCOMT3, CsCOMT18, CsCOMT17, and CsCOMT9, and PtCOMT32, PtCOMT2, PtCOMT5, PtCOMT1, PtCOMT18, and PtCOMT16 (Fig. 7). In summary, the CsCOMT family members undergo large segment duplications within themselves, as well as with other species, meaning that some family members share homologous segments with those from other species.

Fig. 7
figure 7

The collinearity analysis of CsCOMTs. A Large-scale duplication analysis of CsCOMTs; B Large-scale duplication analysis of COMTs between tea plants and Arabidopsis; C Large-scale duplication analysis of COMTs between tea plants and poplar

Full size image

The cis-acting elements predicted to be present in CsCOMTs promoters

To explore the putative biological functions of CsCOMT members, the cis-acting elements in the promoter regions of CsCOMTs were analyzed. As shown in Fig. 8A and B, the CsCOMTs promoters contained various enriched elements, which mainly include four aspects: plant growth and development, light response, stress response, and hormone response. Many CsCOMT genes contain many types of homeopathic elements, such as CsCOMT12, CsCOMT21, CsCOMT7, CsCOMT24, CsCOMT19, etc. Among them, CsCOMT19 contains 5 types related to growth and development (CAAT-box, CAT-box, CCAAT-box, O2-site, and motif I); There are 6 categories related to photoreaction (chs-CMA1a, chs-CMA2a, G-Box, ATCT-motif, AT1-motif, and Box4); There are 6 types related to stress (MYC, W-box, MYB, P-Box, LTR, and WRE3); There is one type of hormone related (TGACG-motif).

Fig. 8
figure 8

Analysis of cis-acting elements in the promoter region of CsCOMTs. (A) The left figure represents the evolutionary lineage of members of the tea plant COMT family, while the right figure represents the composition of homeostatic elements in the tea plant COMT family. (B) Distribution of cis-acting elements with related functions in CsCOMTs family

Full size image

GO functional enrichment analysis

In order to further elucidate the biological functions of the CsCOMT gene family, we conducted GO enrichment analysis on its members (Fig. 9). The members of the COMT family of tea plants are mainly enriched in the flavonol biosynthetic process, lignin biosynthentic process, and metabolism in biological processes; The main enriched cellular components are cycloplasm, nucleus, and plasma membrane; Molecular functions are mainly enriched in buffered O-methyltransferase activity, methyltransferase activity, myricetin 3 '- O-methyltransferase activity, O-methyltransferase activity, protein dimerization activity, and quercetin 3-O-methyltransferase activity.

Fig. 9
figure 9

CsCOMT functional enrichment analysis

Full size image

Transcriptome data analysis

We used previously published transcriptome dataset to analyze the expression patterns of 25 CsCOMTs in different tissues and under different stress conditions. As shown in Fig. 10, some CsCOMTs were not expressed in multiple tissues, such as CsCOMT20 only expressed in flowers, CsCOMT18, CsCOMT9, CsCOMT2, CsCOMT4, CsCOMT1, and CsCOMT17 only expressed in roots. While some CsCOMTs had high expressions in various tissues (stems, young leaves, mature leaves, old leaves, flowers, fruits, and young buds), such as CsCOMT19, CsCOMT3, CsCOMT25, CsCOMT11, CsCOMT6, CsCOMT24, CsCOMT12, CsCOMT22 and CsCOMT18. In addition, some CsCOMTs were also expressed in a few tissues, such as CsCOMT5 in stems, roots, and flowers; CsCOMT14 in young leaves and flowers; CsCOMT13 in old leaves and flowers. These results indicate that different CsCOMT genes may be involved in different growth and developmental processes of tea plants.

Fig. 10
figure 10

Expression of CsCOMTs in different tissues of tea plants

Full size image

Under different stress conditions (salt stress, drought stress), the expression of CsCOMTs were various (Fig. 11). Among them, the expression level of CsCOMT6 gradually decreased with the prolongation of salt stress time, while the expression levels of CsCOMT13 and CsCOMT20 first increased and then decreased. Under drought stress, the expression levels of CsCOMT6 and CsCOMT19 showed a decreasing trend with prolonged drought time, and the expression level of CsCOMT6 decreased more significantly; CsCOMT25 showed an increasing trend with prolonged drought time. Under MeJA treatment, CsCOMT24, CsCOMT7, and CsCOMT10 first decreased and then increased with the prolongation of treatment time, while CsCOMT18 showed an increasing trend.

Fig. 11
figure 11

Expression of CsCOMTs under different stress conditions

Full size image

Expression patterns of CsCOMTs in different tissues organ

We selected six members of the CsCOMT family, including CsCOMT3, CsCOMT5, CsCOMT6, CsCOMT11, CsCOMT19, and CsCOMT25 for qRT-PCR to check the expression patterns in different tissues organ (bud, first leaf, second leaf, third leaf, old leaf, stem, flower, fruit) (Fig. 12). The results showed that CsCOMT3, CsCOMT5, and CsCOMT25 had relatively high expression levels in all plant tissues organ, with the highest expression levels in flowers. CsCOMT6 had relatively high expression levels in old leaves, first leaves, and second leaves; CsCOMT11 had high expression levels in third leaves; and CsCOMT19 had the highest expression level in stems.

Fig. 12
figure 12

Expression of CsCOMTs in different tissues. Different lowercase letters indicate significant differences (P < 0.01)

Full size image

The effect of CsCOMT19 on melatonin content in tea plant

CsCOMT19 has a high expression level in buds and new leaves, and the UTR sequence of this gene is between 300–600 bp, it is suitable for VIGS experiments. Therefore, we constucted CsCOMT19 gene-silenced plants (pTRV2-CsCOMT19) using VIGS method to investigate the effect of down-regulation of CsCOMT19 expression on the accumulation of melatonin in tea plant (Fig. 13A). After verification by qRT-PCR (Fig. 13B), using HPLC to detect the melatonin content in different types of plants (WT, pTRV2, pTRV2-CsCOMT19), it was found that the retention time of the standard sample was 16.530 min (Fig. 13C). In the new buds of WT and pTRV2 plants (control), the melatonin content was 132.17–149.00 ng/g fresh weight (FW) and 117.39–123.12 ng/g FW, respectively. There were no changes in phenotype of CsCOMT19 gene-silenced plants, but the content of melatonin significantly decreased to a range of 64.70–94.29 ng/g FW, with an average decrease of 41% (Fig. 13D). Thus, it can be seen that there was a positive correlation between the CsCOMT19 expression and the content of melatonin, indicating that CsCOMT19 can positively regulate the synthesis of melatonin in the tea plant.

Fig. 13
figure 13

Effects of silencing of CsCOMT19 on melatonin accumulation in tea plant. A Phenotype of CsCOMT19 silenced tea plant; B Chromatogram of melatonin standard (16.530 min); C Content of melatonin in control (WT and pTRV2) and CsCOMT19 silenced plants

Full size image

Discussion

Tea plant is one of the world’s most important beverage crops due to its numerous secondary metabolites conferring tea quality and health effects [24]. The level of stress resistance is an important criterion for evaluating the commercial value of tea plant varieties. The exploration of main-effect genes with higher stress resistance in tea plant has become a research hotspot in tea plant breeding. The COMT gene family has a significant impact on plant growth and resistance to stress [6, 25]. With the continuous development of higher plant genome sequencing, COMT gene family from many plants were identified, which can regulate plant growth and resistance to stress, such as Arabidopsis [11], rice [12], longan [13], etc. However, there are relatively few reports on the COMT in tea plant. Therefore, based on the tea plant genome data, we identified and comprehensively analyzed the members of the tea plant COMT family (CsCOMT).

In this study, a total of 25 CsCOMT members were identified from the tea genome (Table 1, Fig. 3), which is significantly different from other species such as Arabidopsis (17 members) [11], rice (33 members) [12], and populus (7 members) [26]. Most COMT members of tea trees are distributed in the Class I, III, and V families, and they have similar sequences to all members of the COMT family in Arabidopsis, populus and rice (Fig. 1). The COMT family members of tea trees are missing in Class II and Class IV, which may be related to gene replication. Gene duplication probably contributes to the evolution of species and to the adaptation of species to their environments [27]. The results also showed that CsCOMTs were mainly located on chromosome 5. In terms of gene structure, most CsCOMT family members contained few introns (Fig. 4). Studies have shown that contained few introns was more conducive to plant adaptation to environmental changes [28]. The loss of introns in the CsCOMT gene family may be a specific mechanism accompanying evolution, which may promote tea plants to adapt more quickly to stressful environments.

In order to gain a deeper understanding of the evolutionary characteristics of the tea plant’s CsCOMT family, a collinearity analysis was conducted on the tea plant’s own genome. Gene duplication probably contributes to the evolution of species and to the adaptation of species to their environments [27]. Four segments with high duplication were found in the collinearity analysis, which may have been caused by the self-replication of the same genes or by chromosomal structural variations during the evolutionary process (Fig. 7). Based on this, collinearity analysis was performed on tea plant, Arabidopsis, and poplar, revealing 2 and 7 pairs of collinearity, respectively. This result indicates that the CsCOMT family has a higher degree of homology in plants and may have similar biological functions. The analysis of the promoter region cis-acting elements revealed multiple components related to growth and stress resistance (Fig. 8), suggesting that it plays an important role in the growth and stress response of tea plants [29].

The results of transcriptome data analysis indicate that the expression of the tea plant CsCOMT gene family members varies among different parts of the plant, and there are also differences in tissue expression levels among different members (Fig. 10). For example, CsCOMT19, CsCOMT3, CsCOMT25, and CsCOMT11 show high expression in various tissues of the tea plant, and these genes may play a critical regulatory role in tea plant growth. When plants were stressed or treated with external hormones, the expression level of COMTs increased [30,31,32]. The above results indicate that the CsCOMT family is widely involved in non-biological stress responses and growth and development processes in tea plants. In future research, we can focus on these gene members, which is of great significance for exploring their potential biological functions.

Melatonin is an important signaling molecule with various physiological functions in plant responses to abiotic and biotic stresses [33]. COMT involved in melatonin biosynthesis as a key enzyme [10, 11]. In this study, we further investigated the role of CsCOMT19 in melatonin biosynthesis due to qRT-PCR analysis showed that it was expressed in various tissues organ. The results showed that melatonin synthesis decreased significantly after CsCOMT19 silencing, indicating that CsCOMT19 was positively correlation with melatonin content in tea plants.

Conclusions

Here, this study identified and preliminarily predicted the functions of the CsCOMT gene family in tea plant. Our results suggest that the CsCOMTs may play important regulatory roles in tea plant growth and stress response. In addition, CsCOMT19 can positively regulate the synthesis of melatonin in tea plant. This study lays a foundation for exploring the potential applications of the CsCOMTs, provides a basis for in-depth understanding of the biological functions of the CsCOMTs, and offering information for future tea plant breeding.

Materials and methods

Identification and sequence analysis of CsCOMT genes

Using the already identified COMT protein sequences from Arabidopsis and rice as target sequences to search the CsCOMT members from the tea plant genome database (TPIA) by BLAST search tool with an E-value threshold set to be less than 1e-5. Then, the obtained CsCOMT protein sequences were further analyzed for conserved domains using NCBI Conserved Domains Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Expasy website (https://www.expasy.org/resources/compute-pI-mw) was used to analyze the molecular weight, isoelectric point, amino acid length of CsCOMT [34]. The subcellular localization of CsCOMT was predicted by using pattern recognition database (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi) [35]. Large-scale duplication analysis of COMT family members from tea plant, Arabidopsis, and poplar draw circos diagrams was performed using TBtools [36].

Phylogenetic and conserved motif analysis of CsCOMT genes

The phylogenetic tree on CsCOMTs with COMTs from rice, Arabidopsis, and poplar was constructed using MEGA 7.0 software with the neighbor-joining method (Boostrap = 1,000). The gene structure of CsCOMTs were analyzed by GSDS 2.0 (http://gsds.cbi.pku.edu.cn). MEME (http://memesuite.org) was used to analyze the conserved sequences of CsCOMTs. The amino acid polarity and secondary structure of CsCOMTs were predicted by using PSIPRED [37]. SWISS-MODEL was used to predict the CsCOMTs tertiary structure [38]. The 2000 bp sequences of the translation start sites of CsCOMTs was obtained from tea plant genome database, and PlantCARE was applied to predict cis-elements of CsCOMTs [39]. GO enrichment analysis through TPIA database.

Transcriptome data analysis

Based on the identified CsCOMTs, download their transcriptome data that including different tissues (flower, stem, root, apical bud, young leaf, mature leaf, old leaf), salt stress, PEG-induced drought stress, and methyl jasmonate (MeJA) treatment from the TPIA database (http://tpia.teaplant.org). Use TBtools to construct a heatmap for visualization.

Plant materials and sampling

Tea plant ‘Fuding Dabai’ from the germplasm resource nursery of the Tea College at Guizhou University was used as experimental materials. One leaf, two leaves, three leaves, old leaves, buds, flowers, stems, and fruits were harvested and immediately frozen in liquid nitrogen, then stored in -80 ℃ freezer for later use.

VIGS-mediated gene silencing of CsCOMT19 in tea plant

VIGS technology was used to silence of CsCOMT19 in tea plants according to the method previously described [40]. In brief, a 292 bp fragment of CsCOMT19 was assembled into the pTRV2 vector to construct the pTRV2-CsCOMT19 for VIGS. The pTRV1, pTRV2, and pTRV2-CsCOMT19 constructs will then be separately transformed into the Agrobacterium tumefaciens strain GV3101. After cultivation and resuspension, the A. tumefaciens carrying pTRV1 and either pTRV2 or pTRV2-CsCOMT19 were vacuum infiltrated into tea plant cuttings. The inoculated cuttings was kept in the dark for 3 d, then grown in a greenhouse at 25 ℃ with 16 h light / 8 h dark cycle. This study selected 3 empty vector plants and 6 plants with reduced CsCOMT19 gene.

Determination of the melatonin content

Grind the fresh sample and use ultrasonic extraction method. Using a high-performance liquid chromatograph (Waters) with a fluorescent detector, Sunfire C18 column (Waters, 4.6 × 150 mm) was employed. The column was gradient eluted for 18 min with a 42%-55% methanol solution in 0.1% formic acid aqueous solution at a flow rate of 0.6 mL/min with an injection volume of 10 μL. The excitation wavelength was set at 280 nm and the emission wavelength was set at 348 nm. The Agilent 1100 liquid chromatograph was coupled to the 6210 TOF electrospray ionization mass spectrometer with a photodiode array detector. The same column and mobile phase were used, with A being methanol and B being 0.1% formic acid aqueous solution. The gradient elution was as follows: A phase increased from 42 to 55% within 18 min, and then decreased to 42% within 1 min. The flow rate was set at 0.6 mL/min and the column temperature was maintained at 25 ℃. The ionization mode was positive and the nebulizer pressure was set at 45 psi. The drying gas flow rate was set at 12 L/min and the drying gas temperature was set at 35 ℃. The melatonin content was quantified accordinng to the melatonin standard curve, and expressed as ng/g FW.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the harvested samples using the modified CTAB method, then the cDNA was synthesized using the reverse transcription kit (Genenode, Wuhan, China) according to the manufacturer’s instructions. The primers used for gene expression alanysis were designed using Primer Premier 6.0 software (Table S2). The Actin was used as the internal reference gene. qRT-PCR was carried on a CFX100 Realtime PCR System (Bio-Rad, CA, USA). The reaction conditions and system were performed according to the SYBR Green Realtime-PCR Mastermix instructions, and the results were calculated using the relative quantification 2−ΔΔCT method [41]. There are three treatments each time, and each treatment has three plants.

Statistical analysis

Excel 2010 was used to organize and analyze statistical data. The data statistical significance was analyzed by one-way and two-way ANOVA in SPSS 19.0, with the LSD method at the significance level at P < 0.05.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the TPIA database, http://tpia.teaplants.cn/index.html.

References

  1. Tan D, Manchester L, Reiter R, Qi W, Karbownik M, Calvo J. Significance of melatonin in antioxidative defense system: reactions and products. Biol Signals Recept. 2000;9(3–4):137–59.

    Article  PubMed  CAS  Google Scholar 

  2. Rosen J, Than N, Koch D, Poeggeler B, Laatsch H, Hardeland R. Interactions of melatonin and its metabolites with the ABTS cation radical: extension of the radical scavenger cascade and formation of a novel class of oxidation products, C2-substituted 3-indolinones. J Pineal Res. 2006;41(4):374–81.

    Article  PubMed  CAS  Google Scholar 

  3. Tan D, Manchester L, Terron M, Flores L, Reiter R. One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res. 2007;42(1):28–42.

    Article  PubMed  CAS  Google Scholar 

  4. Murch S, Campbell S, Saxena P. The role of serotonin and melatonin in plant morphogenesis: regulation of auxin-induced root organogenesis in in vitro-cultured explants of stJohn’s Wort (Hypericum perforatum L.). In Vitro Cell Dev Biol Plant. 2001;37(6):786–93.

    Article  CAS  Google Scholar 

  5. Wolf K, Kolář J, Witters E, Dongen W, Onckelen H, Macháčková I. Daily profile of melatonin levels in Chenopodium rubrum L. depends on photoperiod. J Plant Physiol. 2001;158(11):1491–3.

    Article  CAS  Google Scholar 

  6. Fan J, Xie Y, Zhang Z, Chen L. Melatonin: a multifunctional factor in plants. Int J Mol Sci. 2018;19(5):1528.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Wang Y, Reiter R, Chan Z. Phytomelatonin: a universal abiotic stress regulator. J Exp Bot. 2017;69(5):963–74.

    Article  Google Scholar 

  8. Xiao S, Liu L, Wang H, Li D, Li C. Exogenous melatonin accelerates seed germination in cotton (Gossypium hirsutum L.). PLoS ONE. 2019;14(6):e0216575.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Back K. Melatonin metabolism, signaling and possible roles in plants. Plant J. 2021;105(2):376–91.

    Article  PubMed  CAS  Google Scholar 

  10. Byeon Y, Lee H, Lee K, Back K. Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating N-acetylserotonin in Arabidopsis. J Pineal Res. 2014;57(2):219–27.

    Article  PubMed  CAS  Google Scholar 

  11. Chen A, Song S, Deng K, Zheng Q, Tan G, Liao H, Zhou J. Genome-wide analysis of COMT gene family in Arabidopsis thaliana. Mol Plant Breed. 2022;20(19):6388–96.

    Google Scholar 

  12. Liang S, Xu S, Qu D, Yang L, Wang J, Liu H, Xin W, Zou D, Zheng H. Identification and functional analysis of the Caffeic Acid O-Methyltransferase (COMT) gene family in rice (Oryza sativa L.). Int J Mol Sci. 2022;23(15):8491.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Xiao X, Liu M, Jiang M, Chen Y, Xue X, Zhou C, Wu X, Wu J, Guo Y, Yeh K, Lai Z, Lin Y. Whole-genome ldentification and expression analysis of SNAT, ASMT and COMT families of melatonin synthesis pathway in Dimocarpus longan. Acta Hortic Sin. 2022;49(05):1031–46.

    Google Scholar 

  14. Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, et al. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Mol Biol. 2003;51(6):973–89.

    Article  PubMed  CAS  Google Scholar 

  15. Yang WJ, Du YT, Zhou YB, Chen J, Xu ZS, Ma YZ, et al. Overexpression of TaCOMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis. Int J Mol Sci. 2019;20(3):652.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Eudes A, Dutta T, Deng K, Jacquet N, Sinha A, Benites VT, et al. SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum. PLoS ONE. 2017;12(6):e0178160.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Cai YC, Zhang KW, Kim H, Hou GC, Zhang XB, Yang HJ, et al. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase. Nat Commun. 2016;7:11989.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Mukhopadhyay M, Mondal T, Chand P. Biotechnological advances in tea (Camellia sinensis (L.) O. Kuntze): a review. Plant Cell Rep. 2016;35(2):255–87.

    Article  PubMed  CAS  Google Scholar 

  19. Liu Y, Wang D, Zhang S, Zhao H. Global expansion strategy of Chinese herbal tea beverage. Adv J Food Sci Technol. 2015;7(9):739–45.

    Article  Google Scholar 

  20. Zaveri N. Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications. Life Sci. 2006;78(18):2073–80.

    Article  PubMed  CAS  Google Scholar 

  21. Lian M, Jiang Y, Lv S, He Y, Zhou J, Meng Q. The anti-obesity effect of instant Pu-erh black tea in mice with hydrogenated oil diet-induced obesity. Appl Mech Mater. 2014;644–650:5248–51.

    Article  Google Scholar 

  22. Cheng Q, Cai S, Ni D, Wang R, Zhou F, Ji B, Chen Y. In vitro antioxidant and pancreatic α- amylase inhibitory activity of isolated fractions from water extract of Qingzhuan tea. J Food Sci Technol. 2015;52(2):928–35.

    Article  PubMed  CAS  Google Scholar 

  23. Chen J, Huang C, Sun C, Wu L, Wu T. Disaster-causing hazard division and evaluation of meteorological disasters for tea in Fujian Province. J Nat Disasters. 2018;27(1):198–207.

    Google Scholar 

  24. Zhang R, Ma Y, Hu X, Chen Y, He X, Wang P, Chen Q, Ho CT, Wan X, Zhang Y, Zhang S. TeaCoN: a database of gene co-expression network for tea plant (Camellia sinensis). BMC Genomics. 2020;21(1):461.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Liu S, Fu C, Gou J, Sun L, Huhman D, Zhang Y, Wang ZY. Simultaneous downregulation of MTHFR and COMT in switchgrass affects plant performance and induces Lesion-Mimic cell death. Front Plant Sci. 2017;8:982.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Barakat A, Choi A, Yassin NB, Park JS, Sun Z, Carlson JE. Comparative genomics and evolutionary analyses of the O-methyltransferase gene family in Populus. Gene. 2011;479(1–2):37–46.

    Article  PubMed  CAS  Google Scholar 

  27. Zhang J. Evolution by gene duplication: an update. Trends Ecol Evol. 2003;18(6):292–8.

    Article  Google Scholar 

  28. Jeffares D, Penkett C, Bahler J. Rapidly regulated genes are intron poor. Trends Genet. 2008;24(8):375–8.

    Article  PubMed  CAS  Google Scholar 

  29. Itoh N, Iwata C, Toda H. Molecular cloning and characterization of a flavonoid-O-methyltransferase with broad substrate specificity and regioselectivity from Citrus depressa. BMC Plant Biol. 2016;16(1):180.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Li W, Lu JX, Lu K, Yuan JL, Huang JH, Du H, et al. Cloning and phylogenetic analysis of Brassica napus L. Caffeic acid O-Methyltransferase 1 gene family and its expression pattern under drought stress. PLoS ONE. 2016;11(11):e0165975.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Fu LP, Xiao YG, Yan J, Liu JD, Wen WE, Zhang Y, et al. Characterization of TaCOMT genes associated with stem lignin content in common wheat and development of a gene-specific marker. J Integr Agric. 2019;18(5):939–47.

    Article  CAS  Google Scholar 

  32. Zhang J, Li Y, Jia HX, Li JB, Huang J, Lu MZ, et al. The heat shock factor gene family in Salix suchowensis: a genome-wide survey and expression profiling during development and abiotic stresses. Front Plant Sci. 2015;6:748.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Qian C, Zhu Q, Gao S, Chen G, Qi S, Ji Z, Jin C, Chen X, Qi X. Effects of exogenous melatonin treatment on cold storage quality and chilling injury of postharvest peach fruit. Jiangsu J Agric Sci. 2020;36(03):702–8.

    Google Scholar 

  34. Wilkins M, Gasteiger E, Bairoch A, Sanchez J, Williams K, Appel R, Hochstrasser D. Protein identification and analysis tools in the ExPASy server. Methods Mol Biol. 1999;112:531–52.

    PubMed  CAS  Google Scholar 

  35. Chou K, Shen H. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS ONE. 2010;5(6):e11335.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13:1194–202.

    Article  PubMed  CAS  Google Scholar 

  37. McGuffin L, Bryson K, Jones D. The PSIPRED protein structure prediction server. Bioinformatics. 2000;16(4):404–5.

    Article  PubMed  CAS  Google Scholar 

  38. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014;42:W252–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Li G, Li Y, Yao X, Lu L. Establishment of a virus-induced gene-silencing (VIGS) system in tea plant and its use in the functional analysis of CsTCS1. Int J Mol Sci. 2022;24:392.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method. 2013;25(4):402–8.

    Article  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by the Natural Science Foundation of China (32260789); Guizhou Province Science and Technology Planning Project (Qiankehe Support [2021] General 111).

Author information

Authors and Affiliations

  1. College of Life Science, The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in the Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025, People’s Republic of China

    Thanh Huyen Pham, Xingyu Tian, Huimin Zhao & Litang Lu

  2. College of Tea Science, Guizhou University, Guiyang, 550025, People’s Republic of China

    Tong Li & Litang Lu

Authors
  1. Thanh Huyen Pham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xingyu Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huimin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Litang Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.T. and L.L. conceived and designed the experiments. P.T. and Z.H. performed the biological assays. P.T. and T.X. performed the bioinformation analysis. P.T. L.T. and L.L. analyzed the data and finalized the manuscript, which was approved by all the authors.

Corresponding authors

Correspondence to Tong Li or Litang Lu.

Ethics declarations

Ethics approval and consent to participate

The study was carried out in compliance with relevant institutional, national, and international quidelines and legislation for plant ethics.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Fig. S1. Number of COMT families in the C.sinensi, A.thaliana, O.sativa and P.trichocarpa, and the proportion of the total number of 104, respectively. Fig. S2. Number of COMT families in the different groups. Fig. S3. The sequences of 15 motifs of CsCOMT in tea plant. Table S1. The distribution ratio of CsCOMT genes on each chromosome in C.sinensis. Table S2. Primer sequence for qRT-PCR.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pham, T.H., Tian, X., Zhao, H. et al. Genome-wide characterization of COMT family and regulatory role of CsCOMT19 in melatonin synthesis in Camellia sinensis. BMC Plant Biol 24, 51 (2024). https://doi.org/10.1186/s12870-023-04702-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-023-04702-0

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us